PROJECT SUMMARY/ABSTRACT The risk of anal cancer in HIV-infected individuals has markedly increased following the advent of anti-retroviral therapy, especially in HIV-infected men who have sex with men (MSM). 90% percent of invasive anal squamous cell carcinomas are associated with human papillomavirus (HPV) infection, of which 80-90% are associated with HPV type 16. Although anal HPV-16 infection is common in HIV-infected MSM, only a small fraction of infected men ever develops anal cancer. Why this occurs in some but not others is unknown. Recent advances in technology have made it feasible to sequence and study the genetic composition, or ?microbiome?, of the community of microbes that colonize different parts of the human body. The expansion of otherwise commensal bacteria within the gastrointestinal tract is postulated to contribute to inflammation and carcinogenesis via a variety of pathways including the production of superoxide radicals and toxins. These imbalances have been associated with disease states such as inflammatory bowel disease and colorectal cancer. This study will determine whether a similar association exists between the anal canal microbiome and high- grade squamous intraepithelial lesions (HSIL), the precursor to invasive anal cancer. We will determine whether enrichment of the anal canal with pro-inflammatory bacteria and depletion of anti-inflammatory bacteria is associated with anal HSIL, particularly because long-term chronic inflammation has been proposed as a key mediator in HPV cancer pathogenesis. Accordingly our specific aims are: 1) To characterize the differences in ? and ?-diversity of the anal microbiome in HIV-infected, anal HPV-16 infected, MSM on effective ART with and without anal HSIL; and 2) To identify specific differences in the composition of the anal microbiome between HIV-infected, anal HPV-16 infected, MSM on effective ART with and without anal HSIL. Our results will contribute to our understanding of HPV carcinogenesis and may potentially provide new approaches to diagnosis and treatment of anal HSIL.